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. 2017 May;91(5):438–450. doi: 10.1124/mol.116.106245

Bardoxolone Methyl and a Related Triterpenoid Downregulate cMyc Expression in Leukemia Cells

Un-Ho Jin 1, Yating Cheng 1, Beiyan Zhou 1,1, Stephen Safe 1,✉
PMCID: PMC5399643  PMID: 28275049

Abstract

Structurally related pentacyclic triterpenoids methyl 2-cyano-3,12-dioxoolean-1,9-dien-28-oate [bardoxolone-methyl (Bar-Me)] and methyl 2-trifluoromethyl-3,11-dioxoolean-1,12-dien-30-oate (CF3DODA-Me) contain 2-cyano-1-en-3-one and 2-trifluoromethyl-1-en-3-one moieties, respectively, in their A-rings and differ in the position of their en-one structures in ring C. Only Bar-Me forms a Michael addition adduct with glutathione (GSH) and inhibits IKKβ phosphorylation. These differences may be due to steric hindrance by the 11-keto group in CF3DODA-Me, which prevents Michael addition by the conjugated en-one in the A-ring. In contrast, both Bar-Me and CF3DODA-Me induce reactive oxygen species in HL-60 and Jurkat leukemia cells, inhibit cell growth, induce apoptosis and differentiation, and decrease expression of specificity proteins (Sp) 1, 3, and 4, and cMyc, and these effects are significantly attenuated after cotreatment with the antioxidant GSH. In contrast to solid tumor–derived cells, cMyc and Sp transcriptions are regulated independently and cMyc plays a more predominant role than Sp transcription factors in regulating HL-60 or Jurkat cell proliferation and differentiation compared with that observed in cells derived from solid tumors.

Introduction

Reactive oxygen species (ROS) play an important role in cellular homeostasis. Increased ROS levels result in oxidative stress and dysregulation of cell growth and survival, and ROS-induced cell damage including oxidative DNA damage may play a role in development of some cancers (Fruehauf and Meyskens, 2007; Trachootham et al., 2009; Hole et al., 2011). Because of the high expression of ROS in cancer cells (Farquhar and Bowen, 2003; Battisti et al., 2008; Kumar et al., 2008; Trachootham et al., 2009; Hole et al., 2011; Chen et al., 2013; Lee et al., 2015; Sriskanthadevan et al., 2015), and particularly in myeloid leukemia cells (Farquhar and Bowen, 2003; Bossis et al., 2014; Sriskanthadevan et al., 2015), ROS inducers such as arsenic trioxide (Miller et al., 2002; Trachootham et al., 2009) can selectively target cancer cells and are highly effective inhibitors of leukemia cell growth and survival (Trachootham et al., 2009; Hole et al., 2011). The triterpenoid methyl 2-cyano-3,12-dioxoolean-1,9-dien-28-oate [bardoxolone-methyl (Bar-Me)] induces ROS and decreases mitochondrial membrane potential in some leukemia cells and in cancer cell lines derived from solid tumors (Ito et al., 2000; Konopleva et al., 2002, 2004b, 2005; Stadheim et al., 2002; Ikeda et al., 2003, 2004; Suh et al., 2003a,b; Ahmad et al., 2006; Samudio et al., 2006, 2008; Yue et al., 2006; Brookes et al., 2007; Liby and Sporn, 2012). Promising objective tumor responses were observed in a phase I human clinical trial with Bar-Me in patients with advanced solid tumors and lymphomas, and according to Hong et al. (2012) the results support “continued development of other synthetic triterpenoids in cancer.”

Methyl 2-cyano-3,11-dioxo-18β-olean-1,12-dien-30-oate (CDODA-Me) is an isomer of Bar-Me (Fig. 1A) and was synthesized from glycyrrhetinic acid, the active component of licorice (Chadalapaka et al., 2008). CDODA-Me and Bar-Me are pentacyclic triterpenoids with the same oleanane skeletal structure and 2-en-3-one moieties in their A-rings; they differ with respect to the position of their E-ring carboxyl groups (C-28, Bar-Me; C-30, CDODA-Me), and the 11-oxo-12-ene functionality in the C-ring of CDODA-Me versus the 9-ene-12-oxo moiety in the C-ring of Bar-Me (Fig. 1A). It has previously been reported that CDODA-Me, a more active 2-trifluoromethyl analog [methyl 2-trifluoromethyl-3,11-dioxoolean-1,12-dien-30-oate (CF3DODA-Me)], and Bar-Me (Fig. 1A) downregulate specificity protein (Sp) transcription factors (TFs) Sp1, Sp3, and Sp4 and pro-oncogenic Sp-regulated genes in breast and thyroid cancer cell lines (Chintharlapalli et al., 2011; Kim et al., 2012), and that Bar-Me-induced downregulation of Sp TFs in pancreatic cancer cells is ROS dependent (Jutooru et al., 2010). In this study, we show that Bar-Me and CF3DODA-Me also induced ROS in HL-60 and Jurkat leukemia cell lines, and that ROS plays an important role in drug-mediated growth inhibition, induction of apoptosis and differentiation, and downregulation of Sp1, Sp3, Sp4, and cMyc. In contrast to solid tumor–derived cells, cMyc does not regulate expression of Sp TFs and plays the more dominant role in maintaining cell proliferation and a decreased differentiation phenotype. We also observed that, unlike Bar-Me, CF3DODA-Me did not alkylate thiol-containing proteins and this may correlate with decreased toxic side effects. Although there are some mechanistic differences in the effects of Bar-Me and CF3DODA-Me in leukemia cells both compounds target downregulation of cMyc and thus like cMyc inhibitors such as BET bromodomain inhibitors, they represent a novel therapeutic strategy for treating leukemia (Delmore et al., 2011).

Fig. 1.

Fig. 1.

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Modulation of thiols by Bar-Me, CDODA-Me, and CF3DODA-Me in leukemia cells. (A) Structures of oleanolic and glycyrrhetinic acid–derived triterpenoids CDDO-Me (Bar-Me), CDODA-Me, and CF3DODA-Me. (B) Spectral changes in Bar-Me, CDODA-Me, and CF3DODA-Me after incubation with GSH. (C) Comparative A-, B-, and C-ring structures of Bar-Me and CDODA-Me/CF3DODA-Me. (D) Cells were treated with dimethylsulfoxide (DMSO), Bar-Me, and CF3DODA-Me for 6 hours, and TNFα was then added for 15 minutes. Whole cell lysates were analyzed by western blots as outlined in Materials and Methods.

Materials and Methods

Cell Lines, Antibodies, and Reagents.

HL-60, Jurkat, and U937 cell lines were obtained from ATCC (Manassas, VA) or kindly provided by Dr. Michael Andreeff, University of Texas MD Anderson Cancer Center (Houston, TX). Cells were maintained in RPMI 1640 (GenDEPOT, Houston, TX) supplemented with 10% fetal bovine serum (FBS) (Sigma-Aldrich, St. Louis, MO) according to ATCC recommendations. Cells were maintained at 37°C in the presence of 5% CO2, and the maximum concentration of solvent (dimethylsulfoxide) used in the experiments was 0.1%. The Sp1 antibody was purchased from Epitomics (Burlingame, CA). The Sp3, Sp4, and EGFR antibodies were purchased from Santa Cruz Biotechnology (Dallas, TX). NFκB Pathway Ab Sampler Kit, phospho-protein kinase B (Akt) (Ser473) Pathway Ab Sampler Kit, p-MAPK Family Ab Sampler Kit, cleaved caspase-3, cleaved caspase-7, cleaved caspase-8, cleaved caspase-9, cleaved poly(ADP-ribose) polymerase (PARP), Met, Survivin, p-GSK3α/β (S21/9), GSK3α/β, p-mTOR (S2448), mTOR, p-p70S6K (T389), p70S6K, p-4E-BP (S65), 4E-BP, and GAPDH antibodies were purchased from Cell Signaling Technology (Danvers, MA). CD11b-FITC and CD4-FITC antibodies for fluorescence-activated cell sorter (FACS) analysis were purchased from Abcam (Cambridge, MA). L-Glutathione reduced and other chemicals in this study were purchased from Sigma-Aldrich. CM-H2DCFDA was purchased from Molecular Probes (Grand Island, NY). MG-132, LY294002, and MK-2206 were purchased from Selleckchem (Houston, TX).

Spectrophotometric Analysis of Interaction between Bar-Me or CF3CDODA-Me and Glutathione (GSH) Reduced.

The reactivity of Bar-Me or CF3CDODA-Me was examined as previously described (Chadalapaka et al., 2008). Briefly, 20 mM Bar-Me and CF3CDODA-Me stock were prepared in polyethylene glycol 200 and 75 mM GSH stock was prepared in assay buffer (100 mM potassium phosphate, pH 6.5, 1 mM EDTA, and 0.1% Triton X-100). Bar-Me or CF3CDODA-Me (16.5 μl; final concentration 660 μM) was combined with GSH (16.5 μl; final concentration 2.5 mM) in 450 µl of assay buffer and water was made up to 500 µl of total reaction volume. The spectra were obtained at room temperature, immediately upon preparation using a Beckman Du 640 spectrophotometer (Fullerton, CA).

Cell Proliferation (XTT) Assay.

The cytotoxic effects of compounds on cells were investigated using a XTT Cell Viability kit (Cell Signaling Technology) according to the manufacturer’s instructions. Briefly, the cells were plated in 96-well culture plates at a density of 2 × 104 cells/well in 100 µl of RPMI medium containing 2.5% FBS and various compounds or other treatments. After 1–3 days of culture, 50 µl of XTT reaction solution was added to each well. The optical density was read at 490 nm wavelength in an enzyme-linked immunosorbent assay plate reader after 4-hour incubation of the plates (37°C and 5% CO2 + 95% air). All determinations were confirmed using replication in at least three identical experiments.

ROS and Mitochondrial Membrane Potential Determinations.

ROS was determined by flow cytometry as previously described (Jutooru et al., 2014). Briefly, cellular ROS levels were evaluated with the cell permeable probe CM-H2DCFDA according to the manufacturer’s protocol (Molecular Probes). Cells were seeded at a cellular density of 1 × 106 cells/ml in 6-well plates, pretreated with GSH for 1 hour, and treated with vehicle (dimethylsulfoxide), Bar-Me, or CF3DODA-Me for 3 hours, and then CM-H2DCFDA (5 μg) was added in 1 ml of treated culture media and incubated for 30 minutes. ROS was determined by flow cytometry as described previously (Kim et al., 2012). The mitochondrial membrane potential was measured using a tetramethylrhodamine mitochondrial membrane potential assay kit (Abcam) according to the manufacturer’s instructions. Briefly, cells were seeded at a cellular density of 1 × 106 cells/ml in 6-well plates, pretreated with GSH for 1 hour, and then treated with vehicle (dimethylsulfoxide), Bar-Me, or CF3DODA-Me for 6 hours. Cells were stained with 100 nM tetramethylrhodamine for 20 minutes. Accumulated tetramethylrhodamine in active mitochondria was determined by flow cytometry (FL2 channel).

Annexin V Staining and Surface Differentiation Marker Detection.

Cells were stained using the Alexa Fluor 488 Annexin V/Dead Cell Apoptosis Kit according to the manufacturer’s protocol (Molecular Probes). To detect the CD11b or CD4 surface antigens, CD11b or CD4 antibodies conjugated with fluorescein isothiocyanate were added to Bar-Me or CF3CDODA-Me-pretreated cells in 1X phosphate-buffered saline containing 3% bovine serum albumin for 1 hour at 4°C. Annexin V and surface marker were analyzed by flow cytometry as described previously (Kim et al., 2012). The patterns of CD11b, CD4, and ROS on the graphs were represented by the positively counted cell numbers divided at the same FL1-A value of 500,000 cells to control.

Quantitative Real-Time Polymerase Chain Reaction (PCR).

Quantitative real-time PCR was determined as previously described (Jutooru et al., 2014). Briefly, cDNA was prepared from the total RNA of cells using the High Capacity RNA-to-cDNA Kit (Applied Biosystems, Foster City, CA). Each PCR was carried out in triplicate in a 20 μl volume using SYBR Green Mastermix (Applied Biosystems, Carlsbad, CA) for 15 minutes at 95°C for initial denaturing, followed by 40 cycles of 95°C for 15 seconds and 60°C for 1 minute in the iCycler (MyiQ2) Real-Time PCR System (Bio-Rad, Hercules, CA). The comparative computed tomography method was used for relative quantitation of samples. Values for each gene were normalized to expression levels of the TATA-binding protein. The sequences of the primers used for real-time PCR were as follows: c-MYC sense 5′-GTC AAG AGG CGA ACA CAC AA-3′, antisense 5′-GGC CTT TTC ATT GTT TTC CA-3′; IL-2 sense 5′-TCA CCA GGA TGC TCA CAT TT-3′, antisense 5′-GCA CTT CCT CCA GAG GTT TG-3′; IL-4 sense 5′-CAG ACA TCT TTG CTG CCT CC-3′, antisense 5′-GTG TCC TTC TCA TGG TGG CT-3′; IL-13 sense 5′-GGT CAA CAT CAC CCA GAA CC-3′, antisense 5′-GAT TCC AGG GCT GCA CAG TA-3′; and TATA-binding protein sense 5′-GAT CAG AAC AAC AGC CTG CC-3′, antisense 5′-TTC TGA ATA GGC TGT GGG GT-3′.

Western Blot Analysis.

Western blot analysis was performed as previously described (Jutooru et al., 2014). Briefly, cells (1 × 106/ml) were plated in the fresh RPMI media containing 2.5% FBS for 1 hour and then treated with different concentrations of the compounds for the indicated times. Cellular lysates were prepared in a lysis buffer containing 50 mM Tris-HCl (pH 7.5), 2 mM ethylenediaminetetraacetic acid, 150 mM NaCl, 0.5% deoxycholate, and 0.1% sodium dodecylsulfate, in each 10 μl/ml protease and phosphatase inhibitor cocktail (GenDEPOT) and 1% NP-40. The cells were disrupted and extracted at 4°C for 30 minutes. After centrifugation, the supernatant was obtained as the cell lysate. Protein concentrations were measured using the Bio-Rad DC protein assay. Aliquots of cellular proteins were electrophoresed on 4%–20% Mini-PROTEAN TGX Gel (Bio-Rad) and transferred to a polyvinylidene difluoride membrane. The membrane was allowed to react with a specific antibody, and detection of specific proteins was carried out by enhanced chemiluminescence. Loading differences were normalized using a polyclonal GAPDH antibody.

Transfection of Small Inhibitory RNA.

Transfection of small inhibitory RNAs was performed using the Neon electroporation system (Invitrogen, Waltham, MA). Cells (5 × 106 cells) were washed with 1X phosphate-buffered saline, resuspended in 100 μl of electroporation buffer, and transfected with 100 nM of each siRNA duplex using the Neon electroporator (Invitrogen) following the manufacturer’s protocol (1350 V, 3× of 10 mm pulses). The electroporated cells were then cultured in RPMI medium containing 10% FBS and incubated for 72 hours. After incubation, the cells were collected for FACS analysis, western blot analysis, XTT assay, or quantitative real-time PCR assay.

Statistics.

All of the experiments were repeated a minimum of three times and the data are expressed as the mean ± S.E.M. Statistical significance was analyzed using the Student’s t test and a P value of less than 0.05 was considered statistically significant.

Results

Bar-Me and CF3DODA-Me Differentially Interact with GSH and IKKβ.

Induction of ROS by 2-cyano-3,12-dioxooleana-1,9-dien-28-oic acid (CDDO) compounds is due, in part, to alkylation of thiol-containing proteins (Ahmad et al., 2006; Yore et al., 2006), and CDDO forms a Michael addition product with GSH at the C-1 position of the A-ring (Couch et al., 2005). Incubation of Bar-Me with GSH resulted in formation of a 290 nm peak (Fig. 1B) as previously described for CDDO (Couch et al., 2005); in contrast, CDODA-Me and CF3DODA-Me did not induce spectral changes associated with a Michael addition product. This may be due to steric hindrance by the 11-keto group, which inhibits alkylation at C-1 (Fig. 1C). CF3DODA-Me was more cytotoxic than CDODA-Me in cancer cells (Chadalapaka et al., 2008), and the comparative effects and mechanisms of action of Bar-Me and CF3DODA-Me were further investigated in HL-60 and Jurkat cells. Tumor necrosis factor (TNF) α–induced phosphorylation of IKKβ in U937 cells was inhibited by Bar-Me, which formed a Cys-179 adduct/IKKβ adduct (Ahmad et al., 2006) and we observed similar results for Bar-Me in U937 and HL-60 cells (phospho-IKKβ was not detected in Jurkat cells) (Fig. 1D). In contrast, CF3DODA-Me did not decrease TNFα-induced expression of phospho-IKKβ in U937 and HL-60 cells, and Bar-Me but not CF3DODA-Me decreased phosphorylation of p65(NFκB) in HL-60 and U937 cells as previously reported for Bar-Me in the latter cell line (Ahmad et al., 2006). These results complement the GSH addition experiments illustrated in Fig. 1B, suggesting that Michael addition–dependent adducts are lower for CF3DODA-Me compared with Bar-Me and this may result in decreased toxicity for the former compound.

Bar-Me and CF3DODA-Me Inhibit Leukemia Cell Growth and Induce Cell Death and Differentiation.

Bar-Me and CF3DODA-Me induced time- and concentration-dependent inhibition of metabolic activity (Fig. 2, A and B) and also induced cleavage of caspase-3, caspase-8, and PARP in HL-60 and Jurkat cells (Fig. 2C). Induction of apoptosis was further confirmed by FACS analysis (Fig. 2D) showing that both compounds induced annexin V staining and increased the percentage of apoptotic cells. Bar-Me and CF3DODA-Me also induced the differentiation marker CD11b in HL-60 cells (Fig. 2E) previously reported for CDDO in HL-60 cells (Konopleva et al., 2004a). Both Bar-Me and CF3DODA-Me induced the surface antigen CD4 in Jurkat cells (Fig. 2F), indicating an increase in cell maturation and differentiation.

Fig. 2.

Fig. 2.

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Bar-Me and CF3DODA-Me inhibit leukemia cell growth and induce apoptosis and differentiation of leukemia cells. HL-60 and Jurkat cells were treated with Bar-Me (A) or CF3DODA-Me (B) and growth inhibition was determined using the XTT assay. HL-60 and Jurkat cells were treated with Bar-Me or CF3DODA-Me for 24 hours and the effects of cleaved PARP and caspases (C), annexin V staining (D), and apoptosis were determined by western blots and FACS analysis for annexin V and propidium iodide staining, respectively, as outlined in Materials and Methods. Cells were treated with dimethylsulfoxide (DMSO), Bar-Me, and CF3DODA-Me for 72 hours and differentiation markers (CD11b) (E) in HL-60 and CD4 in Jurkat (F) cells were determined by FACS analysis as outlined in Materials and Methods. Results are expressed as mean ± S.E.M. for at least three replicate determinations and significant (P < 0.05) changes (compared with DMSO) are indicated (#).

Bar-Me and CF3DODA-Me Induce ROS and ROS-Dependent Anticancer Activities.

The results illustrated in Fig. 3A show that treatment with both Bar-Me and CF3DODA-Me for 3 hours significantly induced ROS in HL-60 cells as determined using FACS analysis and the fluorescent probe CM-H2DFCDA. GSH alone decreased basal ROS and in cotreatment studies GSH inhibited induction of ROS by Bar-Me and CF3DODA-Me in HL-60 cells; moreover, similar results were observed in Jurkat cells (Fig. 3B). We also observed that induction of ROS by Bar-Me and CF3DODA-Me was important for their growth inhibitory effects using the XTT assay since cotreatment with GSH partially reversed this response (Fig. 3C). Bar-Me and CF3DODA treatment of HL-60 and Jurkat cells for 12 hours induced cleavage (activation) of caspases-8, -9, -3, and -7 and PARP cleavage, and we also observed induction of annexin V staining in both cell lines (Supplemental Fig. 1). The apoptotic responses (caspase/PARP cleavage and induction of annexin V) induced by Bar-Me and CF3DODA-Me were significantly blocked after cotreatment with GSH. The results illustrated in Supplemental Fig. 2, A and B, show that these triterpenoids also decreased mitochondrial membrane potential, which was reversed after cotreatment with GSH, and this could be one source of induced ROS production. Previous studies show that Bar-Me induces differentiation in leukemia cells (Konopleva et al., 2004a) and both Bar-Me and CF3DODA-Me induce CD11b mRNA levels in HL-60 cells (Fig. 4A); this response was also inhibited after cotreatment with GSH. Treatment of HL-60 cells with both triterpenoids for 72 hours also induced expression of CD11 cell surface protein as determined by FACS analysis; however, cotreatment with GSH reversed this response for CF3DODA-Me but not for Bar-Me, suggesting that the latter compound also induced ROS-independent differentiation. In Jurkat cells treatment with Bar-Me and CF3DODA-Me for 24 hours induced the percentage of CD4-positive cells and IL2, IL4, and IL13 mRNA levels, and all of these induced responses were attenuated after cotreatment with GSH. Thus, the results in Figs. 3 and 4 demonstrate that Bar-Me/CF3DODA-Me–induced ROS plays an important role in cell proliferation, survival/apoptosis, and differentiation. We also observed that the non-thiol antioxidant Tiron inhibited Bar-Me/CF3DODA-Me–induced ROS, CD11-positive (HL-60) and CD4-positive (Jurkat) cells, and caspase-3 (cleavage); however, Tiron did not block compound-dependent growth inhibition (Supplemental Figs. 3 and 4).

Fig. 3.

Fig. 3.

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Bar-Me and CF3DODA-Me induce ROS in leukemia cells. HL-60 (A) and Jurkat (B) cells were pretreated with GSH for 1 hour, and then treated with dimethylsulfoxide (DMSO), Bar-Me, or CF3DODA-Me for 3 hours. ROS was determined by FACS analysis as outlined in Materials and Methods. (C) Cells were treated with DMSO, Bar-Me, or CF3DODA-Me alone or in combination with GSH for 24 hours and cell proliferation was determined using the XTT assay as outlined in Materials and Methods. (D) Cells were treated with DMSO, Bar-Me, or CF3DODA-Me alone or in combination with GSH for 24 hours, and cleaved PARP and caspases were determined by western blots of whole cell lysates as outlined in Materials and Methods. Results are expressed as mean ± S.E.M. for at least three replicate determinations and significant (P < 0.05) effects of the compounds (#) and reversal of specific effects by GSH (*) are indicated.

Fig. 4.

Fig. 4.

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Bar-Me and CF3DODA-Me induce markers of differentiation and maturation. HL-60 cells were treated with Bar-Me or CF3DODA-Me alone or in combination with GSH and the effects of CD11b mRNA (A) and protein levels (B) were determined after 24 and 72 hours, respectively, as outlined in Materials and Methods. Jurkat cells were treated with Bar-Me or CF3DODA-Me alone or in combination with GSH and CD4 (C) and were determined by FACS analysis after 72 hours and IL-2, IL-4, and IL-13 mRNA after 24-hour levels (D) were determined by real-time PCR as outlined in Materials and Methods. Results are expressed as mean ± S.E.M. (at least three replicate determinations), and significant (P < 0.05) induction by the compound (#) and inhibition by GSH (*) are indicated.

Bar-Me and CF3DODA-Me-Induced ROS: Role of Downregulation of cMyc and Sp TFs and Phosphorylation of Akt.

Bar-Me induced ROS-dependent downregulation of Sp1, Sp3, Sp4, and pro-oncogenic Sp-regulated gene products in pancreatic cancer cells (Jutooru et al., 2010); similar results were observed for both compounds in HL-60 cells (Fig. 5A), whereas in Jurkat cells the effects were less pronounced for Bar-Me and only observed at higher cytotoxic concentrations (data not shown). The effects of Bar-Me and CF3DODA-Me on putative Sp-regulated genes including EGFR, cMET, surviving, and bcl2 were both compound and cell-context dependent. cMet was only expressed in Jurkat cells and was decreased by both compounds; EGFR expression was decreased only by CF3DODA-Me, and expression of survivin and bcl2 was unaffected by both compounds. The pattern of Bar-Me- and CF3DODA-Me-mediated downregulation of Sp-regulated genes in leukemia versus solid tumor–derived cells was different and was cell-context dependent. Bar-Me and CF3DODA-Me induced downregulation of Sp1, Sp3, and Sp4 proteins, and these effects were attenuated in HL-60 and Jurkat cells after cotreatment with GSH (Fig. 5B). We also observed that the superoxide scavenger Tiron attenuated the effects of CF3DODA-Me but not Bar-Me on Sp proteins and cMyc in HL-60 cells (Supplemental Fig. 4C), and these differences are currently being investigated. ROS-dependent downregulation of Sp proteins in solid tumors is due, in part, to rapid epigenetic silencing of cMyc (Jutooru et al., 2014; Hedrick et al., 2015a). Bar-Me and CF3DODA-Me induced a rapid decrease in cMyc protein levels in HL-60 and Jurkat cells (within 3 hours). Sp1, Sp3, and Sp4 expression was also rapidly decreased in HL-60 (but not Jurkat) cells (Fig. 5C) and cotreatment of HL-60 and Jurkat cells with GSH attenuated downregulation of cMyc (Fig. 5D). A recent study reported that activation of PAK-1 (phosphorylation) regulated cMyc expression in leukemia cells (Pandolfi et al., 2015); however, our results showed minimal to nondetectable pPAK1 expression and that Bar-Me and CF3DODA-Me did not alter pPAK-1/PAK-1 expression (data not shown), suggesting that ROS may directly affect cMyc expression. ROS-dependent downregulation of cMyc in solid tumor–derived cells resulted in decreased expression of cMyc-regulated micro-RNAs (miR-27a and miR-20a/miR-17) and increased expression of the miR-regulated transcriptional repressors ZBTB10, ZBTB34, and ZBTB4 (Jutooru et al., 2010, 2014; Safe et al., 2014; Hedrick et al., 2015a); however, ZBTB4 and ZBTB34 were not detectable in HL-60 or Jurkat cells and ZBTB10 expression was decreased by Bar-Me and CF3DODA-Me (Supplemental Fig. 5). Thus, ROS-dependent downregulation of cMyc and Sp TFs in HL-60 and Jurkat cells was not due to disruption of miR:ZBTB interactions, and the mechanism is currently being investigated.

Fig. 5.

Fig. 5.

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Bar-Me and CF3DODA-Me decrease Sp proteins and cMyc in leukemia cells. (A) Cells were treated with dimethylsulfoxide (DMSO), Bar-Me, or CF3DODA-Me for 24 hours and whole cell lysates were analyzed by western blots as outlined in Materials and Methods. (B) Cells were treated with DMSO, Bar-Me, or CF3DODA-Me alone or in combination with GSH for 24 hours and whole cell lysates were analyzed by western blots as outlined in Materials and Methods. (C) Cells were treated with DMSO, Bar-Me, or CF3DODA-Me for 3, 6, or 12 hours and whole cell lysates were analyzed by western blots as outlined in Materials and Methods. (D) Cells were treated with DMSO, Bar-Me, or CF3DODA-Me alone or in combination with GSH for 9 hours and analyzed by western blots as outlined in Materials and Methods, and results are expressed as mean ± S.E.M. for at least three replicate determinations. Significant (P < 0.05) effects of the compounds (#) and reversal by GSH (*) are indicated.

cMyc and Sp TFs regulate multiple pro-oncogenic genes/pathways in solid tumor–derived cell lines; knockdown of Sp1, Sp3, and Sp4 results in decreased cell growth, induction of apoptosis, and decreased cell migration/invasion (Jutooru et al., 2010, 2014; Safe et al., 2014; Hedrick et al., 2015a). The effects of cMyc, Sp1, Sp3, and Sp4 were further investigated by RNA interference; knockdown in HL-60 cells did not affect caspase-3 and PARP cleavage (Fig. 6A). The loss of Sp1, Sp3, or Sp4 had minimal effects on HL-60 cell proliferation (only Sp4 knockdown showed some activity), whereas knockdown of cMyc resulted in decreased cell growth (Fig. 6B). The loss of cMyc also resulted in the induction of CD11b mRNA levels and HL-60 cells expressing CD11b, whereas depletion of Sp1, Sp3, and Sp4 had minimal effects (Fig. 6C). Parallel experiments were carried out in Jurkat cells (Fig. 6, D–G), where knockdown of cMyc, Sp1, Sp3, and Sp4 did not affect caspase-3 and PARP cleavage and only sicMyc and siSp3 decreased cell growth (Fig. 6E). Knockdown of cMyc in Jurkat cells also increased CD4, IL-2, IL-4, or IL-3; siSp3 also increased the percentage of CD4-positive cells and siSp1, siSp3, and siSp4 increased expression of IL-2 in Jurkat cells. Thus, Bar-Me and CF3DODA-Me induce similar ROS-dependent inhibition of cell growth and induction of differentiation in HL-60 and Jurkat cells. This is primarily due to downregulation of c-Myc with only some effects due to decreased expression of Sp TFs.

Fig. 6.

Fig. 6.

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Role of cMyc and Sp TFs in leukemia cell growth, differentiation, and maturation. Knockdown of Sp1 (siSp1), Sp3 (siSp3), Sp4 (siSp4), all three Sp TFs (siSp1/3/4), and cMyc (sicMyc) in HL-60 cells was carried out by RNA interference, and the effects on protein expression (A) and cell proliferation (B) were determined by western blots and the XTT assay. Effects of knockdown on CD11b mRNA and cellular expression of CD11b protein (C) were determined by real-time PCR and FACS analysis, respectively. Jurkat cells were transfected with siSp1, siSp3, siSp4, siSp1/3/4, and sicMyc, and effects on protein levels (D), cell growth (E), CD4-positive cells (F), and IL-2, IL-4, and IL-3 mRNA levels (G) were determined by western blots, XTT assay, FACS analysis, and real-time PCR, respectively, as outlined in Materials and Methods. Results are expressed as mean ± S.E.M. (three replicate determinations) and significant (P < 0.05) effects compared with dimethylsulfoxide control are indicated (#).

Previous studies have shown that in solid tumor–derived cell lines, ROS inducers inhibit mTOR signaling through activation of sestrin 2; however, treatment of HL-60 and Jurkat cells with Bar-Me or CF3DODA-Me did not induce sestrin 2 (data not shown). However, the results in Fig. 7A show that in HL-60 and Jurkat cells, Bar-Me and CF3DODA-Me decreased phosphorylation (activation) of mTOR, Akt, and GSK3α/β (downstream from Akt). This was due, in part, to a decrease in total Akt and mTOR proteins, which was partially reversed by cotreatment with GSH (Fig. 7A). The effects of GSH were more pronounced in Jurkat cells treated with CF3DODA-Me. We also examined the time course effects of Bar-Me and CF3DODA-Me on phosphokinases in HL-60 and Jurkat cells, which showed a similar pattern of responses in these cell lines (Supplemental Fig. 6). Both Bar-Me and CF3DODA-Me decreased phosphorylation of Akt, mTOR, p70S6K, and 4E-BP1 in HL-60 cells; however, with the exception of 4E-BP1, the decreased phosphorylation correlated with decreased total protein. In Jurkat cells, Bar-Me and CF3DODA-Me decreased p-Akt (after 12 hours) but not total protein, mTOR phosphorylation or total protein was not affected, and p70S6K (total and phosphoprotein) and 4E-BP1 (phosphoprotein) were also decreased. The contributions of Bar-Me/CF3DODA-Me–dependent inhibition of Akt activation were further investigated using the PI3K/Akt inhibitors LY294002 and MK2206. Both inhibitors blocked Akt, p70S6K, and 4E-BP1 but not mTOR activation in HL-60 and Jurkat cells (Fig. 7B). Moreover, these inhibitors also inhibited cMyc expression. The possible linkage between Bar-Me/CF3DODA-Me–mediated inhibition of the PI3K/Akt pathway and cMyc downregulation is currently being investigated.

Fig. 7.

Fig. 7.

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Inactivation of mTOR and Akt by Bar-Me and CF3DODA-Me. (A) Cells were treated with Bar-Me or CF3DODA-Me alone or in combination with 5 mM GSH for 12 hours and analyzed by western blots. Cells were treated with LY294002 (PI3K) and MK2208 (Akt) kinase inhibitors for 24 hours, and whole cell lysates were analyzed by western blots (B) by determining the effects on cell growth in HL-60 and Jurkat cells (C) and on CD11b (HL-60 cells) (D) and CD4 (Jurkat cells) (E) differentiation markers as outlined in Materials and Methods. Results (C–E) are mean ± S.E.M. for at least three replicate determinations and significant (P < 0.05) differences compared with dimethylsulfoxide (DMSO) (control) are indicated (#).

Discussion

In this study, Bar-Me and CF3DODA-Me, a structurally related triterpenoid, were used as model ROS-inducing agents to investigate their mechanisms of cytotoxicity in HL-60 and Jurkat cells. In addition, since clinical applications of Bar-Me have been associated with some toxic side effects (de Zeeuw et al., 2013), we also investigated the effects of structural differences in the C-ring of Bar-Me and CF3DODA-Me on protein thiol adduct formation, a marker of potential toxicity (Fig. 1). Several studies have shown that Bar-Me alkylates thiol groups to form adducts, and using GSH as a model the adduct at C-1 was identified (Couch et al., 2005) and characterized as the expected Michael addition product, which was observed in the A-ring of Bar-Me but not in the C-ring that also contains a conjugated enone (Fig. 1, A and B). GSH adduct formation with Bar-Me can be observed spectroscopically by an increased peak at approximately 290 nm (Fig. 1B) (Couch et al., 2005); however, incubation of GSH with CF3DODA-Me or CDODA-Me did not increase absorption at 290 nm, suggesting the lack of substantial adduct formation. Bar-Me forms an adduct with IKKβ through interactions with Cys-179, resulting in the inhibition of p-IKKβ (Ahmad et al., 2006), and Bar-Me inhibited TNFα-induced phosphorylation of IKKβ in U937 and HL-60 cells as previously reported in U937 cells (Ahmad et al., 2006); however, CF3DODA-Me did not inhibit TNFα-induced p-IKKβ, suggesting the lack of adduct formation and inactivation of IKKβ (Fig. 1D). As illustrated in Fig. 1C, the C-1 position of CDODA-Me, which is important for alkylation via Michael addition, may be sterically hindered by the C-11 ketone moiety compared with Bar-Me, where the ketone is at C-12. The lack of Michael addition activity and subsequent adduct formation may decrease the toxic side effects the CF3DODA-Me analogs compared with Bar-Me (de Zeeuw et al., 2013), and this will be investigated in future studies.

Bar-Me and CF3DODA-Me decrease expression of Sp1, Sp3, and Sp4 in cell lines derived from solid tumors (Jutooru et al., 2010; Chintharlapalli et al., 2011) and Bar-Me induces ROS-dependent downregulation of Sp TFs in pancreatic cancer cells (Jutooru et al., 2010). Subsequent studies have shown that ROS-dependent downregulation of Sp TFs in solid tumor–derived cell lines is due to an initial epigenetic mechanism that involves downregulation of cMyc (O’Hagan et al., 2011; Jutooru et al., 2014). This is followed by decreased expression of cMyc-regulated miRs (miR-27a, miR-20a, and miR-15), resulting in the induction of miR-regulated Sp-repressors ZBTB10, ZBTB34, and ZBTB4 that compete for binding at guanine-cytosine–rich Sp binding sites in promoters of Sp-regulated genes (Jutooru et al., 2014; Hedrick et al., 2015b). These studies showed that many of the cMyc-induced responses including cell proliferation, survival, and migration/invasion were primarily due to regulation of Sp TFs, which were characterized as nononcogene addiction genes (Hedrick et al., 2016). Both Bar-Me and CF3DODA-Me also induced ROS in HL-60 and Jurkat cells, and one of the objectives of this study was to focus on the differences (Fig. 1) and similarities of Bar-Me and CF3DODA-Me in leukemia cell lines. In addition, this research focused on the similarities and differences between solid tumor–derived cell lines versus leukemia cells with respect to the ROS-Myc-miR-ZBTB-Sp pathway.

With the exception of the alkylation of GSH and IKKβ by Bar-Me and not CF3DODA-Me (Fig. 1), most of the effects of both compounds were quantitatively comparable including the ROS-dependent inhibition of cell growth and induction of apoptosis, differentiation, and related genes. These compounds also induced similar ROS-dependent responses in solid tumor–derived cancer cell lines; however, the mechanisms were strikingly different. Bar-Me and CF3DODA-Me induced ROS-dependent downregulation of cMyc and Sp1, Sp3, and Sp4 (Fig. 5, B and D); however, this was not accompanied by epigenetic silencing of the cMyc promoter (data not shown) or modulation of the miR-ZBTB axis (Supplemental Fig. 5) as was observed in solid tumor–derived cells. The lack of a cMyc-Sp connection was confirmed by RNA interference studies (Fig. 6, A and D), which showed that knockdown of cMyc did not affect expression of Sp1, Sp3, and Sp4 and knockdown of Sp TFs did not affect cMyc expression. These results highlight the major differences in ROS-dependent effects in leukemia cell lines versus solid tumor–derived cells.

It was also evident from the results of RNA interference studies that cMyc played a more predominate role in HL-60 and Jurkat cell proliferation and differentiation compared with Sp TFs, and that neither Sp TFs nor cMyc regulated survival pathways. In contrast, it was reported that Sp1, Sp3, and Sp4 were dominant factors in regulating cell growth and survival in solid tumor–derived cells, particularly since cMyc regulated expression of these nononcogene addiction genes (Jutooru et al., 2014; Hedrick et al., 2015a, 2016, 2017).

We did not observe ROS-dependent induction of sestrin-2 in leukemia cells as was previously observed in cells derived from solid tumors (Hedrick et al., 2015a,b; 2017); however, both Bar-Me and CF3DODA-Me differentially affected mTOR and Akt activation and the latter pathway was mTOR independent (Liu et al., 2013; Nawroth et al., 2011) and more predominant in Jurkat cells (Fig. 7). The drug-induced activation of ROS resulted in decreased c-Myc expression (Fig. 5D); Myc was also decreased after inhibition of PI3K/Akt (Fig. 7, A and B). The mechanisms of the compound-dependent inhibition of cMyc and Akt phosphorylation and their interactions are currently being investigated.

In summary, this study shows that Bar-Me and CF3DODA-Me induce ROS, resulting in inhibition of leukemia cell growth and survival and induction of differentiation. Although Bar-Me and CF3DODA-Me are oleanolane-derived pentacyclic triterpenoids, our results show that CF3DODA-Me exhibits decreased thiol alkylation and this may correlate with decreased toxic side effects. Pretreatment of cells with GSH partially reversed Bar-Me- and CF3-DODA-Me-induced cell growth inhibition, induction of apoptosis, CD11-positive (HL-60) and CD4-positive (Jurkat) cells, and decreased cMyc expression. Due to concerns regarding possible GSH adduct formation with the triterpenoids, we carried out parallel experiments with the non-thiol antioxidant Tiron. The effects were similar to that observed for GSH but with two exceptions: Tiron did not reverse compound-induced growth inhibition and it did not reverse Bar-Me-induced downregulation of cMyc (Supplemental Figs. 3 and 4). Despite the mechanistic differences between the action of Bar-Me and CF3DODA-Me on cMyc, both compounds downregulated cMyc expression in leukemia cells. This has potential clinical applications since BET bromodomain inhibitors that target cMyc are also effective inhibitors of leukemia (Delmore et al., 2011; Zuber et al., 2011).

Abbreviations

Akt

protein kinase B

Bar-Me

bardoxolone-methyl (methyl 2-cyano-3,12-dioxoolean-1,9-dien-28-oate)

CDDO

2-cyano-3,12-dioxooleana-1,9-dien-28-oic acid

CDODA-Me

methyl 2-cyano-3,11-dioxo-18β-olean-1,12-dien-30-oate

CF3DODA-Me

methyl 2-trifluoromethyl-3,11-dioxoolean-1,12-dien-30-oate

FACS

fluorescence-activated cell sorter

FBS

fetal bovine serum

GSH

glutathione

PARP

poly(ADP-ribose) polymerase

PCR

polymerase chain reaction

ROS

reactive oxygen species

Sp

specificity protein

TF

transcription factor

TNF

tumor necrosis factor

Authorship Contributions

Participated in research design: Jin, Zhou, Safe.

Conducted experiments: Jin, Cheng.

Contributed new reagents or analytic tools: Safe.

Performed data analysis: Jin, Cheng.

Wrote or contributed to the writing of the manuscript: Jin, Safe.

Footnotes

This work was supported by the National Institutes of Health National Institute of Environmental Health Sciences [Grant P30-ES023512]; Texas AgriLife Research; and the Sid Kyle Endowment.

Inline graphicThis article has supplemental material available at molpharm.aspetjournals.org.

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